EP3874276B1 - Procédé et système d'identification et de quantification d'une protéine - Google Patents

Procédé et système d'identification et de quantification d'une protéine Download PDF

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EP3874276B1
EP3874276B1 EP19805100.5A EP19805100A EP3874276B1 EP 3874276 B1 EP3874276 B1 EP 3874276B1 EP 19805100 A EP19805100 A EP 19805100A EP 3874276 B1 EP3874276 B1 EP 3874276B1
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Prior art keywords
impurity
protein
homodimer
mixed
sec
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EP3874276A1 (fr
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Yuetian Yan
Shunhai WANG
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph

Definitions

  • the invention generally pertains to a method of quantifying a protein.
  • the method for quantifying an impurity in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase with a flow rate of 0.2 ml/min to 0.4 ml/min.
  • the method for quantifying an impurity can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the impurity can be a degradation product of a protein.
  • This disclosure at least in part, provides a method for detecting an impurity in a sample.
  • the method for detecting an impurity in a sample can comprise contacting said sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with a charge-charge interaction functionality.
  • the method for detecting an impurity in a sample can comprise contacting about 10 ⁇ g to about 100 ⁇ g of a sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality.
  • the method for detecting an impurity in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase to provide an eluent including the impurity.
  • the method for detecting an impurity in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase that can be compatible with a mass spectrometer.
  • the method for detecting an impurity in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase, wherein the mobile phase can be selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof.
  • the method for detecting an impurity in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase containing up to 600 mM total salt concentration.
  • the method for detecting an impurity in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase with a flow rate of 0.2 ml/min to 0.4 ml/min.
  • the method for detecting an impurity can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the impurity can be a product-related impurity.
  • the method for detecting an impurity can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the impurity can be a digestion product of a protein.
  • the method for detecting an impurity can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the impurity can be a post-translational modification of a protein.
  • the method for detecting an impurity can comprise detecting an amount of the impurity in eluent using a mass spectrometer, wherein the mass spectrometer can be a tandem mass spectrometer.
  • the method for detecting an impurity can comprise detecting an amount of the impurity in said eluent using a mass spectrometer, wherein the mass spectrometer can be a native mass spectrometer.
  • This disclosure at least in part, provides a method for detecting and/or quantifying a target protein in a sample.
  • the method for detecting and/or quantifying a target protein in a sample can comprise contacting said sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with a hydrophobic interaction functionality
  • the method for detecting and/or quantifying a target protein in a sample can comprise contacting said sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with a charge-charge interaction functionality.
  • the method for detecting and/or quantifying a target protein in a sample can comprise contacting about 10 ⁇ g to about 100 ⁇ g of a sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality.
  • the method for detecting and/or quantifying a target protein in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase to provide an eluent including the impurity.
  • the method for detecting and/or quantifying a target protein in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase that can be compatible with a mass spectrometer.
  • the method for detecting and/or quantifying a target protein in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase, wherein the mobile phase can be selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof.
  • the method for detecting and/or quantifying a target protein in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase containing up to 600 mM total salt concentration.
  • the method for detecting and/or quantifying a target protein in a sample can comprise washing the mixed-mode size exclusion chromatography resin using a mobile phase with a flow rate of 0.2 ml/min to 0.4 ml/min.
  • the method for detecting and/or quantifying a target protein can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the target protein can be an antibody.
  • the method for detecting and/or quantifying a target protein can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the target protein can be a bispecific antibody.
  • the method for detecting and/or quantifying a target protein can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the target protein can be a therapeutic protein.
  • the method for detecting and/or quantifying a target protein can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the target protein can be a product-related impurity of a biopharmaceutical process of manufacturing a protein.
  • the method for detecting and/or quantifying a target protein can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the target protein can be a degradation product of a protein.
  • the method for detecting and/or quantifying a target protein can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the target protein can be a digestion product of a protein.
  • the method for detecting and/or quantifying a target protein can comprise contacting the sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the target protein can be a homodimer species of a multispecific antibody product.
  • the method for detecting and/or quantifying a target protein can comprise quantifying an amount of the target protein in said eluent using a mass spectrometer, wherein the mass spectrometer can be a tandem mass spectrometer.
  • the method for detecting and/or quantifying a target protein can comprise quantifying an amount of the target protein in said eluent using a mass spectrometer, wherein the mass spectrometer can be a native mass spectrometer.
  • This disclosure at least in part, provides a mixed-mode chromatographic system a chromatographic column having a mixed-mode size exclusion chromatography resin with an additional functionality and a mass spectrometer.
  • the mixed-mode chromatographic system can comprise a mixed-mode size exclusion chromatography resin with hydrophobic interaction functionality.
  • the mixed-mode chromatographic system can comprise a mixed-mode size exclusion chromatography resin with charge-charge interaction functionality.
  • the mixed-mode chromatographic system can comprise a mixed-mode size exclusion chromatography resin with an additional functionality which can be used for elution of about 10 ⁇ g to about 100 ⁇ g of a sample.
  • the mixed-mode chromatographic system can comprise a mixed-mode size exclusion chromatography resin capable of receiving a mobile phase.
  • the mixed-mode chromatographic system can comprise a mixed-mode size exclusion chromatography resin capable of being washed with a mobile phase.
  • the mixed-mode chromatographic system can comprise a mass spectrometer coupled to a chromatographic column having a mixed-mode size exclusion chromatography resin with an additional functionality.
  • the mixed-mode chromatographic system can comprise a tandem mass spectrometer.
  • the mixed-mode chromatographic system can comprise a native spectrometer.
  • Impurities in biopharmaceuticals can cause changes that could potentially impact the efficacy, clearance, safety, and immunogenicity of the desired product.
  • oxidation of methionine and tryptophan side chains can affect antibody binding to Fc receptors and antigens ( Bertolotti-Ciarlet et al. Mol. Immunol. (2009) 46: 1878-1882 ; Pan et al. Protein Sci. (2009) 18: 424-433 ; Wei et al. Anal. Chem. (2007) 79: 2797-2805 ; and Wang et al. Mol. Immunol. (2011) 48: 860-866 ).
  • Biopharmaceutical products are required to show high levels of potency, purity, and low level of structural heterogeneity. Structural heterogeneity often affects the bioactivity and efficacy of a drug. Therefore, characterizing and quantifying the therapeutic protein and/or the impurities is important in pharmaceutical drug development. Structural heterogeneity in a protein can arise from post-translational modifications as well as inherent chemical modifications during manufacturing and storage conditions. For proteins produced in the biotechnology industry, complementary separation techniques can be necessary both to purify the target protein and to give an accurate picture of the quality of the final product. The complexity of the product eliminates the use of simple one-dimensional separation strategies. Therefore, an accurate and efficient method of detecting and/or quantifying the therapeutic protein and/or impurities is needed.
  • the disclosure provides a method for quantifying and/or detecting a protein and/or an impurity in a sample.
  • protein includes any amino acid polymer having covalently linked amide bonds. Proteins comprise one or more amino acid polymer chains, generally known in the art as “polypeptides". “Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. “Synthetic peptides or polypeptides' refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
  • a protein may contain one or multiple polypeptides to form a single functioning biomolecule.
  • a protein can include any of bio-therapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies.
  • a protein can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like.
  • Proteins may be produced using recombinant cell-based production systems, such as the insect bacculovirus system, yeast systems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives like CHO-K1 cells).
  • yeast systems e.g., Pichia sp.
  • mammalian systems e.g., CHO cells and CHO derivatives like CHO-K1 cells.
  • proteins comprise modifications, adducts, and other covalently linked moieties.
  • adducts and moieties include for example avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitope, fluorescent labels and other dyes, and the like.
  • avidin streptavidin
  • biotin glycans
  • glycans e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides
  • PEG polyhistidine
  • FLAGtag maltose binding protein
  • Proteins can be classified on the basis of compositions and solubility and can thus include simple proteins, such as, globular proteins and fibrous proteins; conjugated proteins, such as, nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as, primary derived proteins and secondary derived proteins.
  • the protein can be an antibody, a bispecific antibody, a multispecific antibody, antibody fragment, monoclonal antibody, or combinations thereof.
  • antibody includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, C H 1, C H 2 and C H 3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region.
  • the light chain constant region comprises one domain (C L 1).
  • V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of the anti-big-ET-1 antibody may be identical to the human germline sequences, or may be naturally or artificially modified.
  • An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
  • antibody also includes antigen-binding fragments of full antibody molecules.
  • antigen-binding portion of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • an "antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody.
  • antibody fragments include, but are not limited to, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv fragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd' fragment, a Fd fragment, and an isolated complementarity determining region (CDR) region, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multi specific antibodies formed from antibody fragments.
  • CDR complementarity determining region
  • Fv fragments are the combination of the variable regions of the immunoglobulin heavy and light chains, and ScFv proteins are recombinant single chain polypeptide molecules in which immunoglobulin light and heavy chain variable regions are connected by a peptide linker.
  • an antibody fragment contains sufficient amino acid sequence of the parent antibody of which it is a fragment that it binds to the same antigen as does the parent antibody; in some exemplary embodiments, a fragment binds to the antigen with a comparable affinity to that of the parent antibody and/or competes with the parent antibody for binding to the antigen.
  • An antibody fragment may be produced by any means.
  • an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence.
  • an antibody fragment may be wholly or partially synthetically produced.
  • An antibody fragment may optionally comprise a single chain antibody fragment.
  • an antibody fragment may comprise multiple chains that are linked together, for example, by disulfide linkages.
  • An antibody fragment may optionally comprise a multi-molecular complex.
  • a functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids.
  • bispecific antibody includes an antibody capable of selectively binding two or more epitopes.
  • Bispecific antibodies generally comprise two different heavy chains, with each heavy chain specifically binding a different epitope-either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa.
  • the epitopes recognized by the bispecific antibody can be on the same or a different target (e.g., on the same or a different protein).
  • Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen.
  • nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.
  • a typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by a C H 1 domain, a hinge, a C H 2 domain, and a C H 3 domain, and an immunoglobulin light chain that either does not confer antigen-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding or one or both of the heavy chains to one or both epitopes.
  • BsAbs can be divided into two major classes, those bearing an Fc region (IgG-like) and those lacking an Fc region, the latter normally being smaller than the IgG and IgG-like bispecific molecules comprising an Fc.
  • the IgG-like bsAbs can have different formats, such as, but not limited to triomab, knobs into holes IgG (kih IgG), crossMab, orth-Fab IgG, Dualvariable domains Ig (DVD-Ig), Two-in-one or dual action Fab (DAF), IgG-single-chain Fv (IgGscFv), or ⁇ -bodies.
  • the non-IgG-like different formats include Tandem scFvs, Diabody format, Single-chain diabody, tandem diabodies (TandAbs), Dual-affinity retargeting molecule (DART), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock (DNL) method.
  • TandAbs Dual-affinity retargeting molecule
  • DART-Fc Dual-affinity retargeting molecule
  • nanobodies or antibodies produced by the dock-and-lock (DNL) method.
  • DNL dock-and-lock
  • BsAbs are not limited to quadroma technology based on the somatic fusion of two different hybridoma cell lines, chemical conjugation, which involves chemical cross-linkers, and genetic approaches utilizing recombinant DNA technology.
  • Examples of bsAbs include those disclosed in the following patent applications: U.S. Pat. No. 8,586,713, filed June 25, 2010 ; U.S. Pat. Publication No. 2013/0045492, filed June 5, 2012 ; U.S. Pat. No. 9,657,102, filed September 19, 2013 ; U.S. Pat. Publication No. 2016/0024147, filed July 24, 2015 ; U.S. Pat. Publication No. 2018/0112001, filed September 22, 2017 ; U.S. Pat. Publication No.
  • the IEX method utilizes a gradient elution, which will likely compromise MS-based quantitation due to different ionization efficiency of antibodies eluting under different solvent conditions (e.g., pH or salt concentrations).
  • multispecific antibody refers to an antibody with binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibody and KIH Trispecific can also be addressed by the system and method disclosed herein.
  • Certain post-translational modifications involve the addition of other proteins or peptides, such as ISGylation (covalent linkage to the ISG15 protein (Interferon-Stimulated Gene)), SLTMOylation (covalent linkage to the SUMO protein (Small Ubiquitin-related MOdifier)) and ubiquitination (covalent linkage to the protein ubiquitin).
  • ISGylation covalent linkage to the ISG15 protein (Interferon-Stimulated Gene)
  • SLTMOylation covalent linkage to the SUMO protein (Small Ubiquitin-related MOdifier)
  • ubiquitination covalent linkage to the protein ubiquitin
  • mass spectrometer includes a device capable of detecting specific molecular species and measuring their accurate masses.
  • the term is meant to include any molecular detector into which a polypeptide or peptide may be eluted for detection and/or characterization.
  • a mass spectrometer can include three major parts: the ion source, the mass analyzer, and the detector.
  • the role of the ion source is to create gas phase ions. Analyte atoms, molecules, or clusters can be transferred into gas phase and ionized either concurrently (as in electrospray ionization). The choice of ion source depends heavily on the application.
  • mass analyzer includes a device that can separate species, that is, atoms, molecules, or clusters, according to their mass.
  • mass analyzers that could be employed for fast protein sequencing are time-of-flight (TOF), magnetic / electric sector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR), and also the technique of accelerator mass spectrometry (AMS).
  • TOF time-of-flight
  • Q quadrupole mass filter
  • QIT quadrupole ion trap
  • FTICR Fourier transform ion cyclotron resonance
  • AMS accelerator mass spectrometry
  • the mobile phase can have a flow rate of about 0.1 ml/min to about 0.4 ml/min. In one exemplary embodiment, the flow rate of the mobile phase can be about 0.1 ml/min, about 0.15 ml/min, about 0.20 ml/min, about 0.25 ml/min, about 0.30 ml/min, about 0.35 ml/min, or about 0.4 ml/min.
  • the target protein can be a protein with a pI in the range of about 4.5 to about 9.0.
  • the target protein can be a product-related impurity.
  • the product related impurity can be molecular variants, precursors, degradation products, fragmented protein, digested product, aggregates, post-translational modification form, or combinations thereof.
  • the target protein can be a process-related impurity.
  • the process-related impurity can include impurities derived from the manufacturing process, i.e., nucleic acids and host cell proteins, antibiotics, serum, other media components, enzymes, chemical and biochemical processing reagents, inorganic salts, solvents, carriers, ligands, and other leachables used in the manufacturing process.
  • the number of impurities in the sample can be at least two.
  • the post-translationally modified protein can be formed on oxidation of a protein.
  • the target protein can include a degradation product.
  • the degradation product can include a post-translation modification of a therapeutic protein.
  • washing the mixed-mode chromatography resin using a mobile phase requires less than about 30 minutes.
  • the time required for washing the mixed-mode chromatography resin using a mobile phase can be about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, or about 30 minutes.
  • the chromatographic system can be used for at least about 3 sample runs without cleaning. In one aspect, the chromatographic system can be used for at least about 3 sample runs, at least about 4 sample runs, at least about 5 sample runs, at least about 6 sample runs, at least about 7 sample runs, or at least about 8 sample runs, without cleaning.
  • a mixed-mode chromatographic system comprising a chromatographic column 110 capable of being washed using a mobile phase to provide an eluent including a target protein and a mass spectrometer 120 coupled to the chromatographic column (as illustrated in FIG. 4 ).
  • the chromatographic column 110 can be capable of being contacted with a sample using a sample loading device 100.
  • the chromatographic column 110 can be capable of being washed with a mobile phase.
  • the mobile phase can be ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof.
  • the total concentration of the mobile phase that can be used with the chromatographic column 110 can range up to about 600 mM.
  • the total concentration of the mobile phase that can be used with the chromatographic column 110 can be about 5 mM, about 6 mM, 7 mM, about 8 mM, 9 mM, about 10 mM, 12.5 mM, about 15 mM, 17.5 mM, about 20 mM, 25 mM, about 30 mM, 35 mM, about 40 mM, 45 mM, about 50 mM, 55 mM, about 60 mM, 65 mM, about 70 mM, 75 mM, about 80 mM, 75 mM, about 95 mM, 100 mM, about 1100mM, 120 mM, about 130 mM, 140 mM, about 150 mM, 160 mM, about 170 mM, 180 mM, about 190 mM, 200 mM, about 225 mM, 250 mM, about 275 mM, 300 mM, about 325 mM, 350
  • the chromatographic column 110 can be capable of being coupled with a mass spectrometer 120.
  • the mass spectrometer 120 can comprise a nano-spray.
  • the mass spectrometer 120 can be a tandem mass spectrometer.
  • the mass spectrometer 120 can be a native mass spectrometer.
  • the mixed-mode chromatographic system can be capable of detection 140 and/or quantification 130 of a target protein (See FIG. 4 ). In one aspect, the mixed-mode chromatographic system can be used for detection 140 and/or quantification 130 of one target protein.
  • the mixed-mode chromatographic system can be capable of detection 140 and/or quantification 130 of a monoclonal antibody.
  • the mixed-mode chromatographic system can be capable of detection 140 and/or quantification 130 of a therapeutic antibody.
  • the mixed-mode chromatographic system can be capable of detection 140 and/or quantification 130 of an antibody fragment formed on digestion of the antibody.
  • the mixed-mode chromatographic system can be capable of detection 140 and/or quantification 130 of a target protein, which can be a post-translationally modified protein.
  • the mixed-mode chromatographic system can be capable of detection 140 and/or quantification 130 of a target protein, which can be a degradation product of a protein.
  • the mixed-mode chromatographic system can be capable of detection 140 and/or quantification 130 of a target protein which can be an impurity found in a biopharmaceutical product.
  • the mixed-mode chromatographic system can be capable of detection 140 and/or quantification 130 of a target protein which can be an impurity found during the manufacture of the biopharmaceutical product.
  • the mixed-mode chromatographic system can be capable of detection 140 and/or quantification 130 of a target protein which can be a product-related impurity.
  • the product related impurity can be molecular variants, precursors, degradation products, fragmented protein, digested product, aggregates, post-translational modification form, or combinations thereof.
  • the mixed-mode chromatographic system can be capable of detection 140 and/or quantification 130 of a target protein which can be a process-related impurity.
  • the process-related impurity can include impurities derived from the manufacturing process, i.e., nucleic acids and host cell proteins, antibiotics, serum, other media components, enzymes, chemical and biochemical processing reagents, inorganic salts, solvents, carriers, ligands, and other leachables used in the manufacturing process.
  • the number of impurities in the sample can be at least two.
  • both the purified BsAb and the corresponding homodimer standards were buffer exchanged into 50 mM of Tris-HCl buffer (pH 7.5) and each adjusted to 6 ⁇ g/ ⁇ L based on concentrations determined by Nanodrop (Thermo Fisher Scientific, Bremen, Germany). Subsequently, the bsAb and the two corresponding homodimers were mixed at a ratio of 1:1:1. Finally, sequential dilutions were performed using a 2 ⁇ g/ ⁇ L bsAb solution to prepare a series of spike-in standards with the homodimer levels ranging from 0.1% to 10%.
  • MM-SEC-MS Method Mixed-mode size exclusion chromatography was performed on a Waters I-Class UPLC system equipped with photodiode array (PDA) detector (Waters, Milford, MA, US).
  • PDA photodiode array
  • an isocratic elution method was run for 24 minutes.
  • a post-column splitter (-200:1 ratio) was applied after the SEC separation to reduce the flow to ⁇ 1 ⁇ L/min for nano-ESI-MS analysis, while diverting the remaining high flow to the PDA detector for UV monitoring at 280 nm.
  • a disposable PicoTip Emitter (non-coated, tip: 10 ⁇ 1 ⁇ m) (New Objective, Inc., Woburn, MA, US) was used to achieve nano-ESI.
  • silica surface derivatization such as short alkyl chains or linkage of functional groups
  • the residual silanol groups from a modern SEC column can be effectively shielded, and therefore, dramatically reduce the presence of electrostatic interactions.
  • those newly introduced chemical groups might also result in other enhanced secondary interactions (e.g., hydrophobic interaction) with the protein analyte, as reported in recent studies (Yang et al, 2015, supra; Yan He et al., On-line coupling of size exclusion chromatography with mixed-mode liquid chromatography for comprehensive profiling of biopharmaceutical drug product, 1262 JOURNAL OF CHROMATOGRAPHY A 122-129 (2012 )).
  • This group of molecules maintained a relatively unchanged retention time at varying salt concentrations (30 mM to 300 mM). In this case, as the salt concentration increased, the increase in hydrophobic interaction was likely close to and counteracting the decrease in electrostatic interaction, leading to little shift in retention time.
  • the sample was prepared using the methodology illustrated in example 3.
  • the bsAb4 mixture demonstrated that improved separation could not be achieved by decreasing the salt concentration from 300 mM to 75 mM. This is likely because the three molecules all have near neutral pIs (Table 3), and thus exhibit similar retention behavior with corresponding salt concentration changes. Although further lowering the salt concentration to enhance electrostatic interaction may improve the separation, severe peak tailing will likely occur, thus compromising the quantitation. It is also interesting to note that the elution profile of the bsAb4 mixture broadened as the salt concentration increased.

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Claims (14)

  1. Procédé de quantification d'une impureté dans un échantillon, ledit procédé comprenant :
    la mise en contact dudit échantillon avec un système chromatographique comportant une résine de chromatographie d'exclusion de taille à mode mixte avec une fonctionnalité supplémentaire qui conduit à une séparation à mode mixte ;
    le lavage de ladite résine de chromatographie d'exclusion de taille à mode mixte au moyen d'une phase mobile pour fournir un éluant comprenant l'impureté ; et
    la quantification d'une quantité de l'impureté dans ledit éluant au moyen d'un spectromètre de masse,
    dans lequel le spectromètre de masse est couplé au système chromatographique.
  2. Procédé selon la revendication 1, dans lequel la phase mobile utilisée pour éluer l'impureté comporte de l'acétate d'ammonium, du bicarbonate d'ammonium ou du formiate d'ammonium, ou des combinaisons de ceux-ci.
  3. Procédé selon la revendication 1, dans lequel la phase mobile utilisée pour éluer l'impureté a une concentration totale inférieure à environ 600 mM d'acétate d'ammonium et de bicarbonate d'ammonium.
  4. Procédé selon la revendication 1, dans lequel la phase mobile utilisée pour éluer l'impureté a un débit d'environ 0,2 ml/min à environ 0,4 ml/min.
  5. Procédé selon la revendication 1, dans lequel la quantité de l'échantillon chargé sur la résine de chromatographie d'exclusion de taille à mode mixte est d'environ 10 µg à environ 100 µg.
  6. Procédé selon la revendication 1, dans lequel l'impureté est une impureté apparentée au produit.
  7. Procédé selon la revendication 1, dans lequel l'impureté est un homodimère.
  8. Procédé selon la revendication 1, dans lequel l'échantillon comprend l'impureté et au moins une protéine cible qui sont séparées lorsqu'elles sont éluées.
  9. Procédé selon la revendication 8, dans lequel la protéine cible est un anticorps.
  10. Procédé selon la revendication 9, dans lequel l'anticorps est un anticorps bispécifique.
  11. Procédé selon la revendication 8, dans lequel la protéine cible est un anticorps thérapeutique.
  12. Procédé selon la revendication 1, dans lequel la fonctionnalité supplémentaire est une fonctionnalité d'interaction hydrophobe.
  13. Procédé selon la revendication 1, dans lequel la fonctionnalité supplémentaire est une fonctionnalité d'interaction charge-charge.
  14. Procédé selon la revendication 1, dans lequel le spectromètre de masse est un spectromètre de masse natif.
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